WO2015152146A1 - Procédé de culture de cellules souches embryonnaires haploïdes - Google Patents

Procédé de culture de cellules souches embryonnaires haploïdes Download PDF

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WO2015152146A1
WO2015152146A1 PCT/JP2015/059901 JP2015059901W WO2015152146A1 WO 2015152146 A1 WO2015152146 A1 WO 2015152146A1 JP 2015059901 W JP2015059901 W JP 2015059901W WO 2015152146 A1 WO2015152146 A1 WO 2015152146A1
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cells
haploid
embryonic stem
cell
stem cells
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史敏 石野
知英 李
沙央里 ▲高▼橋
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国立大学法人東京医科歯科大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the present invention relates to a method for culturing haploid embryonic stem cells, and more particularly, to a method for culturing haploid embryonic stem cells while maintaining the monoploidy.
  • the present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method.
  • the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells.
  • Mammals usually have a diploid genome derived from their father and mother. This has the significance that when a disease such as an autosomal recessive genetic disease occurs, the genome derived from one parent does not become a fatal symptom by supplementing the function. However, when performing forward genetics, if it is diploid, it is difficult to directly analyze the relationship between the genotype and the phenotype.
  • Haploid (haploid) cell lines have only one pair of chromosomes, and since genotypes and phenotypes can be seen directly, they are expected to be applied to forward genetics research.
  • Establishment of a haploid embryonic stem cell line has been reported.
  • a haploid embryo is produced by chemically stimulating a mouse unfertilized egg, and an ES cell line is established therefrom, whereby a haploid embryonic cell derived from a female parthenogenetic embryo ( It has been reported that female haploid ES cells, parthenogenetic haploid ES cells, phES cells, phESC) were obtained (Non-Patent Documents 1 to 4).
  • haploid ES cells chromosome doubling (diploidization) occurs naturally, and it is difficult to culture while maintaining the haploid state (monoploidy). Therefore, in order to maintain the haploidity of haploid ES cells, it is necessary to regularly collect cells in the G1 phase of haploid cells (1N cells) using a flow cytometer (FACS). There is.
  • haploid ES cells when applying haploid ES cells to forward genetics research, as described above, there is a need for a method of stably culturing the cells in a haploid for a long period of time, as well as normal ES cells ( Similarly, diploid ES cells established from embryos obtained by natural mating) are also required to have stable properties as pluripotent stem cells. That is, it is necessary to show a gene expression profile similar to that of normal ES cells and to have differentiation pluripotency and high proliferation ability (self-replication ability). Furthermore, naturally, when applied to forward genetics research that searches for a causative gene from a phenotype, it is possible to prevent mutations such as deletion and amplification in genomic DNA. Desired.
  • the present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a method capable of stably culturing haploid ES cells over a long period of time.
  • the present inventors have found that diploid ES cells naturally diploid because the transition from the G2 phase to the M phase of the cell cycle is insufficient.
  • the cell cycle is regularly rotated from G1 phase, S phase, G2 phase, and M phase (see the upper part of FIG. 1), but when haploid cells diploid, G1 It is expected that the cell cycle has failed, such as the period, S period, G2 period, G1 period, S period, G2 period, and M period.
  • DNA synthesis occurs without passing through cell division by passing the S phase twice before entering the M phase in this way, and genomic DNA is doubling in haploid cells. (See the middle part of FIG. 1).
  • Cdc2 is a factor responsible for the transition from the G2 phase to the M phase in the progression of the cell cycle. That is, by culturing haploid ES cells in the presence of inhibitors against Wee1 and Myt1, which are factors that inactivate Cdc2, Cdc2 was constantly activated in the cells. As a result, diploidization of haploid ES cells was suppressed, and culturing was possible for a long time while maintaining monoploidy.
  • haploid ES cells cultured by this method maintain undifferentiated properties, have multipotency in vivo, and can contribute to the formation of chimeric mice. Furthermore, it was also found that the haploid ES cells can be transferred to the germline in chimeric mice.
  • haploid ES cells cultured by this method can be differentiated into Epiblast Stem cells (EpiSC) while maintaining their haploidity, and even in the differentiated cells, the haploidy can be differentiated. It has also been found that it can be maintained, and the present invention has been completed. That is, the present invention relates to a method for culturing haploid embryonic stem cells. The present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method. Furthermore, the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells. More specifically, the present invention provides the following inventions.
  • EpiSC Epiblast Stem cells
  • a method for culturing haploid embryonic stem cells comprising culturing the cells under conditions that constantly activate Cdc2.
  • (2) A method for producing a non-human chimeric animal (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo; (Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
  • the non-human chimeric animal is a non-human germ line chimeric animal whose germ cells are derived from the haploid embryonic stem cells.
  • a method for producing haploid differentiated cells (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) a step of inducing differentiation of the haploid embryonic stem cells. (5) A drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells, comprising a Cdc2 activator as an active ingredient.
  • haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability.
  • differentiated cells can be obtained from such haploid ES cells while maintaining their haploidity, and the diploidy can also be maintained in the differentiated cells. It becomes possible.
  • FIG. 1 It is a schematic diagram showing cell cycle progression assumed in haploid embryonic stem (ES) cells. That is, if normal cell cycle progression is carried out in haploid ES cells, as shown in the upper part of FIG. 1, the haploidity is maintained and the cells are divided and proliferated. Become. However, since haploid ES cells naturally diploidize, for example, as shown in the middle part of FIG. 1, G1 phase, S phase, G2 phase, G1 phase, S phase, G2 phase, As in the M phase, it is assumed that the cell cycle is broken and diploidization occurs.
  • FIG. 2 is a schematic diagram showing that it was possible to suppress somaticization (see the lower part of FIG. 1) and succeeded in culturing for a long time while maintaining monoploidy. It is the schematic which shows the process of collect
  • results of FACS analysis of haploid ES cells (phES-B6GFP-3) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS. Furthermore, it is a histogram showing the results of FACS analysis of haploid ES cells after three subcultures in the presence of PD166285 (see the right in the figure). Results of FACS analysis of haploid ES cells (phES-B6GFP) subcultured 8 times in the absence of PD166285 (see the left in the figure), and 1N cells were sorted from the cells by FACS.
  • Results of FACS analysis of haploid ES cells (phES-B6GFP-4) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS.
  • the upper left panel is a photograph showing the results of observing the appearance after 30 days of immunodeficient mice transplanted subcutaneously with haploid ES cells.
  • the upper right panel is a photomicrograph showing endothelium epithelium tissue observed by HE staining of teratoma extracted from the mouse.
  • the lower left panel is a photomicrograph showing the epidermis tissue observed by HE staining of teratoma extracted from the mouse.
  • the lower right panel is a photomicrograph showing a cartilage tissue observed by HE staining of teratoma extracted from the mouse.
  • FIG. 6 is a Scatcher plot diagram showing the result of comprehensive analysis of the difference in gene expression level with ES cells (mES-B6-ave) established from the obtained embryos using a microarray.
  • “1-3”, “1-4”, “2-1”, “2-2”, “1-5”, and “2-3” on the horizontal axis represent chimera fetus individuals.
  • “Body” indicates the chimera contribution ratio of all remaining parts excluding the head and internal organs from the chimeric individual.
  • Total indicates the chimera contribution ratio (chimera contribution ratio in all tissues) obtained by analyzing the whole individual by grinding it without removing the organ from the chimera individual.
  • the bar at the right end indicates the average value of the ratio of GFP positive cells of 1-3, 1-4, 2-1, 2-2 individuals. It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. . It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. .
  • mESCs indicates the analysis results of mouse ES cells established from embryos obtained by natural mating
  • mEpiSCs indicates the analysis results of epiblast stem cells obtained by differentiating the mouse ES cells.
  • PhESCs indicates the analysis result of haploid ES cells obtained by culturing according to the method of the present invention
  • phEpiSCsP3 indicates epiblast stem cells obtained by differentiating the haploid ES cells.
  • the analysis result of (passage number 3) is shown, “phEpiSCsP5” shows the analysis result of epiblast stem cells (passage number 5) obtained by differentiating the haploid ES cells, and “MEF” is mouse embryo 13
  • the analysis result of the fibroblast obtained from the day embryo is shown, and “H 2 O” shows the result of analyzing water (no template DNA) (negative control).
  • the notation on the left side of the figure indicates the analyzed marker genes (Oct4, Nanog, Rex1, Fgf5), “GAPDH” indicates a positive control in this analysis, and “RT ( ⁇ )” indicates a negative control (reverse transcription). Results without reaction).
  • passage number here means the passage number after differentiating phESC into phEpiSC.
  • the notation on the right side of the figure indicates that each marker gene shown on the left side of the figure is an ES cell-specific marker gene (indicated as “ES”) and an epiblast stem cell-specific marker gene (“EpiSC”). And a marker gene whose expression is recognized in ES cells and epiblast stem cells (denoted as “ES / EpiSC”).
  • the present invention provides a method for culturing haploid embryonic stem cells, the method comprising culturing the cells under conditions that constitutively activate Cdc2.
  • the transition from the G2 phase to the M phase is performed in the progression of the cell cycle. It is possible to promote and maintain the monoploidy. Furthermore, in the haploid embryonic stem cells obtained by culturing in this way, mutations such as deletion and amplification do not occur in the genomic DNA, and the pluripotency and high proliferation ability remain maintained.
  • haploid embryonic stem cell means an embryonic stem cell (ES cell) established from an early embryo that has developed as a haploid, and is described in, for example, Non-Patent Documents 1 to 4.
  • Female parthenogenetic embryo-derived haploid ES cells female haploid ES cells, parthenogenetic haploid ES cells, which are established from the early embryos obtained by generating activated unfertilized eggs as described above PhES cells, phESC).
  • Non-Patent Documents 5 and 6 male parthenogenesis established from early embryos obtained by transplanting, activating and generating sperm nuclei into enucleated unfertilized eggs Examples also include embryos derived from haploid ES cells (male haploid ES cells, androgenetic haploid ES cells, ahES cells, ahESC).
  • the origin of unfertilized eggs and sperm used for establishment of these haploid ES cells is not particularly limited, and examples thereof include humans and non-human animals.
  • non-human animals include rodents such as mice and rats, cows, horses, pigs, sheep, monkeys, dogs, mammals such as cats, and birds such as chickens.
  • any method for activating the unfertilized egg any method may be used as long as the second polar body is released from the egg. For example, a method of chemically stimulating with strontium, ethanol, or the like, The method of giving is mentioned.
  • examples of the early embryo prepared by generating activated unfertilized eggs and used for establishing haploid ES cells include, for example, the 8-cell stage, 16-cell stage, morula stage, or blastocyst
  • the embryo in the stage is mentioned.
  • embryos in the blastocyst stage are preferable from the viewpoint of high ES cell establishment efficiency.
  • they are produced from a pure mouse such as C57BL / 6, they are in the 16 cell stage or morula stage. Embryos are preferred, and embryos in the morula stage are more preferred (see Table 1 below).
  • Examples of the “haploid ES cells” used in the culture method of the present invention include not only naturally derived cells but also cells that have been genetically modified, such as phESC-B6GFP used in the examples described later ( Genetically modified cells).
  • the genetically modified cell may be a cell into which a gene encoding a protein to be expressed is introduced exogenously, such as GFP in the phESC-B6GFP, and may be obtained by a knockout method, an RNA method, an antisense method, etc.
  • a cell in which the function of a specific gene is suppressed may be used, or a cell in which the function of a gene is suppressed or activated randomly.
  • Examples of cells whose gene functions are randomly suppressed or activated include cells treated with chemical mutagens such as EMU, EMS, NMU, and NTG, and DNA-cleaving enzymes such as zinc finger nuclease and TALEN. Cells, cells irradiated with fast neutron rays, gamma rays or ion beams, and cells in which transposons are randomly inserted into genomic DNA. Further, such gene modification may be performed after the establishment of haploid cells, for example, at the individual stage of collecting the unfertilized egg or the sperm before the establishment. It may be a thing.
  • Constant activation of Cdc2 means that Cdc2 is always in a state capable of promoting cell division. That is, in normal cell cycle progression, it means that Cdc2 is in a state where Cdc2 can promote cell division, not limited to G2 to M phase where Cdc2 is activated.
  • Cdc2 In the control of the cell cycle, Cdc2 is inactivated and the transition to M phase is suppressed by phosphorylation of 14th threonine or 15th tyrosine of Cdc2 by Wee1 or Myt1 (in G2 phase) Will stop).
  • Cdc2 is activated by Cdc25, whereby Cdc2 is activated by dephosphorylating the 14th threonine and 15th tyrosine of Cdc2.
  • a condition for activating Cdc2 constantly includes a condition under which phosphorylation of Cdc2 at 14th threonine or 15th tyrosine is constantly suppressed.
  • Such suppression of phosphorylation can be achieved by suppressing the activity of Wee1 or Myt1, enhancing the activity of Cdc25, etc.
  • Wee1 it is preferable to suppress the activity of Myt1, and it is more preferable to suppress both the activities of Wee1 and Myt1.
  • “activity suppression” means not only complete suppression (inhibition) of the activity but also partial suppression. Moreover, suppression or enhancement of the activity of these proteins can also be achieved by suppression or enhancement of the expression of the protein. Therefore, “inhibition or enhancement of activity” in the present invention includes cases achieved through inhibition or enhancement of expression. Furthermore, the suppression or enhancement of expression may be at the transcription level or at the translation level.
  • the constant suppression of phosphorylation of Cdc2 at 14th threonine or 15th tyrosine can be achieved by adding a Cdc2 activator described later to a medium in which haploid embryonic stem cells are cultured, etc. This can be achieved by introducing the activator into cells.
  • the haploid embryonic stem cells in the medium supplemented with PD166285 are used in the culture method of the present invention. Is preferably cultured.
  • the concentration of PD166285 added to the medium is not particularly limited as long as Cdc2 can be constantly activated. From the viewpoint of culturing without causing cell death in haploid embryonic stem cells and the like, 300 nM The following is preferable.
  • the haploid embryonic stem cells may be cultured under the conditions where Cdc2 is constantly activated as described above, and other culture conditions can be determined by those skilled in the art. If it exists, the culture
  • a medium used for culturing haploid embryonic stem cells can be prepared based on a basal medium for culturing known embryonic stem cells.
  • basal media include DMEM medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, ⁇ MEM medium, Ham medium, RPMI 1640 medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improved medium, Examples include IMDM medium, medium 199 medium, and any mixed medium.
  • the medium used for culturing haploid embryonic stem cells may be a serum-containing medium or a serum-free medium.
  • a serum-free medium means a medium that does not contain unprepared or unpurified serum, and may include a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors). From the viewpoint of preventing contamination with components derived from different animals, the serum is preferably derived from the same species as the stem cells.
  • the medium used for culturing haploid embryonic stem cells may or may not contain a serum substitute. Examples of serum substitutes include commercially available products such as knockout serum substitutes (KSR, manufactured by Invitrogen), chemical lipid concentrates (Chemically-defined Lipid concentrated, manufactured by Gibco), and glutamax (produced by Gibco). It is done.
  • a medium used for culturing haploid embryonic stem cells is added with a compound that suppresses differentiation of embryonic stem cells and the like.
  • Such compounds include leukemia inhibitory factor (LIF), MEK inhibitor (PD0325901, AZD6244, CI-1040 (PD184352), RDEA119 (BAY869766), SL327, U0126, PD98059, U0124, U0125), GSK3 inhibitor (CHIR99021, SB216763).
  • TGF ⁇ receptor inhibitors SB431542, etc.
  • FGF receptor inhibitors SU5402, PD1733074, etc.
  • ROCK inhibitors GSK26962A, Y-27632, H-1152, etc.
  • the medium used for the culture of haploid embryonic stem cells includes amino acids (L-glutamine, non-essential amino acids, etc.), reducing agents (2-mercaptoethanol), antibiotics (penicillin, streptomycin, etc.), fatty acids or lipids. , Sugars, vitamins, growth factors, cytokines, antioxidants, organic acids (pyruvic acid, lactic acid, etc.), buffers, inorganic salts, and the like.
  • incubator used for culturing haploid embryonic stem cells known ones used for culturing embryonic stem cells and the like can be used as appropriate, and cell-adhesive incubators (for example, ECM, Matrigel) , A culture vessel coated with a cell support substrate such as gelatin or collagen) or a non-cell-adhesive culture vessel.
  • cell-adhesive incubators for example, ECM, Matrigel
  • non-cell-adhesive culture vessel for example, ECM, Matrigel
  • culture conditions for haploid embryonic stem cells can be appropriately selected and adjusted by those skilled in the art in accordance with known culture conditions for embryonic stem cells.
  • the culture temperature is particularly limited. However, it is usually about 30 to 40 ° C, preferably about 37 ° C.
  • concentration of CO 2 is usually about 1-10%, preferably about 2-5%.
  • the oxygen concentration is usually 1 to 10%.
  • the culture of haploid embryonic stem cells may be an adhesion culture or a non-adhesion culture.
  • haploid embryonic stem cells can also be cultured in the presence of feeder cells.
  • the feeder cells are not particularly limited.
  • mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation (gamma rays or the like) or treatment with antibiotics (mitomycin C or the like) are used.
  • non-adherent culture include dispersion culture, aggregated suspension culture, and suspension culture on a carrier.
  • haploid embryonic stem cells cultured using the above-described culture method are not only maintained monoploidy but also pluripotent without causing deletion or the like in genomic DNA.
  • a chimeric animal can be obtained from the haploid embryonic stem cells.
  • the present invention is a method for producing a non-human chimeric animal comprising: (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo; (Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
  • step (i) is as described in ⁇ Method for culturing haploid embryonic stem cells> above.
  • the “early embryo” into which the haploid embryonic stem cells are introduced may be any non-human animal, such as rodents such as mice and rats, cows, horses, pigs, Examples include sheep, monkeys, dogs, mammals such as cats, and birds such as chickens.
  • the origin of the early embryo is preferably the same (same type) as that of the introduced haploid embryonic stem cell, but may be of a heterogeneous relationship.
  • the early embryonic development stage examples include the 8-cell stage, the 16-cell stage, the morula stage, or the blastocyst stage.
  • an early embryo in the blastocyst stage is used for producing a chimeric embryo.
  • a tetraploid embryo may be used as an early embryo into which a haploid embryonic stem cell is introduced. This is because an animal derived from the haploid embryonic stem cell can be directly obtained by generating a tetraploid chimeric embryo into which the haploid embryonic stem cell has been introduced (A. Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8424-8428).
  • the tetraploid embryo should be prepared by a method known to those skilled in the art, such as electrofusion of blastocysts or electrofusion of 2-cell blastocysts by applying an electric pulse in a mannitol solution. Can do.
  • the “introduction” from the haploid embryonic stem cells to the aforementioned early embryo can be performed using a known chimeric embryo production method such as a microinjection method or an aggregation method.
  • step (iii) the chimeric embryo obtained as described above is transplanted into the maternal womb (uterus or fallopian tube) of the non-human animal and generated, whereby a non-human chimeric animal can be obtained.
  • the non-human animal to which the chimeric embryo is transplanted is preferably a pseudopregnant animal, and is preferably the same animal as the origin of the early embryo.
  • the present invention can also provide a method for producing a non-human germline chimeric animal, wherein the germ cells are derived from haploid embryonic stem cells, comprising the steps (i) to (iii).
  • a non-human germline chimeric animal obtained by this method is mated with a normal animal (wild-type animal) or the non-human germline chimeric animal, and a gene derived from the haploid embryonic stem cell is generated from its offspring. If an individual to be possessed is selected, an animal having a gene derived from the haploid embryonic stem cell (an animal derived from the haploid embryonic stem cell) can also be obtained. For selection of an individual having a gene derived from the haploid embryonic stem cell, various traits can be used as an index. For example, body color or the like can be selected as an index. It is also possible to select by extracting DNA from a part of the body and performing Southern blot analysis or PCR analysis.
  • a genetically modified cell into which the above-mentioned specific foreign gene is introduced is used as the haploid embryonic stem cell introduced into the early embryo
  • an animal having the introduced foreign gene can be obtained.
  • a gene-deficient heterozygous animal can be obtained by using a genetically modified cell in which the function of the specific gene is suppressed.
  • gene-deficient homozygous animals can also be obtained by mating the obtained gene-deficient heterozygous animals.
  • the haploid embryonic stem cell introduced into the early embryo if a genetically modified cell whose gene function is randomly suppressed or activated is used, the phenotype of the obtained non-human chimeric animal or its offspring Based on the above, it is possible to search for a gene that causes the disease (a gene whose function is suppressed or activated). That is, the method of the present invention can be suitably used for forward genetics.
  • the present invention is a method for producing haploid differentiated cells comprising: (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; And (ii) a step of inducing differentiation of the haploid embryonic stem cell.
  • the step (i) is as described in ⁇ Method for culturing haploid embryonic stem cells> above.
  • the differentiation induction in the step (ii) is preferably not performed under conditions that constitutively activate Cdc2 from the viewpoint of not suppressing the differentiation of haploid embryonic stem cells.
  • differentiation induction may be performed by in vitro culture or by in vivo culture. A person skilled in the art can perform a known induction method from a universal stem cell such as an embryonic stem cell to a target differentiated cell. It can be performed by selecting appropriately.
  • the above-described haploid embryonic stem cells are cultured in a non-adhered state by removing factors that suppress the differentiation of cells such as LIF from the above-described haploid embryonic stem cell medium.
  • an embryoid body (EB) containing a tissue differentiated into three germ layers can be formed.
  • this EB is cultured on a substrate such as laminin or fibronectin using a medium supplemented with a differentiation inducer such as retinoic acid or activin to obtain cells differentiated into nervous system cells or blood cells. be able to.
  • target differentiated cells can be directly obtained from the haploid embryonic stem cells by a combination of cytokines, growth factors and the like as described in Examples below. It can also be obtained.
  • the differentiated cells obtained in this way may be stem cells (such as epiblast stem cells) as shown in Examples described later, or may be cells obtained by further differentiation induction from the stem cells.
  • a teratoma formation method as shown in Examples described later can also be used. That is, by transplanting the haploid embryonic stem cells into an immunodeficient animal, a teratoma can be formed in the animal, and a wide variety of differentiated cells can be isolated from the teratoma.
  • the differentiated cells thus obtained can be cultured under conditions that constantly activate Cdc2, whereby the monoploidy of the differentiated cells can be maintained.
  • the conditions for activation are as described above.
  • Such culture can be performed by those skilled in the art by appropriately selecting a medium, temperature, CO 2 concentration and the like suitable for the differentiated cells to be cultured.
  • Cdc2 ⁇ Drug to maintain monoploidy>
  • the present invention provides a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells containing a Cdc2 activator as an active ingredient.
  • the “Cdc2 activator” of the present invention is a compound having an action of suppressing the activity of Wee1 or Myt1, which is a factor inhibiting Cdc2 activation, and more specifically, Wee1 as shown in Examples described later.
  • a low molecular weight compound that binds to Myt1 and RNA that binds to a transcription product of a gene encoding Wee1 or Myt1.
  • Cdc2 activator of the present invention In order to act on cells, it does not require complicated and damaging work such as gene introduction using viral vectors, introduction by electroporation, introduction using transfection reagents, etc., and it is added to the medium From the standpoint that Cdc2 of the cell can be constitutively activated alone, a low molecular weight compound that binds to Wee1 or Myt1 is preferred as the Cdc2 activator of the present invention.
  • Examples of the “low molecular compound having an action of suppressing the activity of Wee1 or Myt1” include PD166285 (2-[[4- [2- (diethylamino) ethoxy] phenyl] amino] -6- (2,6-dichlorophenyl).
  • a low molecular weight compound having an action of suppressing the activity of Wee1 International Publication
  • the low molecular weight compounds having the action of suppressing the activity of Myt1 described in 2000/33842 and physiologically acceptable salts or solvates of these compounds can be mentioned, but both the activities of Wee1 and Myt1 can be suppressed.
  • PD166285 is preferable, and PD166285 dihydrochloride is more preferable.
  • RNA that binds to the transcript of the gene encoding Wee1 or Myt1 may be any RNA that targets Wee1 or Myt1 and can suppress the activity of these proteins through suppression of its expression.
  • examples include dsRNA (double stranded RNA) such as siRNA and shRNA (short haipin RNA) complementary to a transcription product of a gene encoding Myt1.
  • the dsRNA chain length is not particularly limited as long as it can suppress the expression of the target gene and does not exhibit toxicity, and is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs. More preferably, it is 21 to 30 base pairs.
  • the dsRNA need not be completely identical to the base sequence of the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more of sequence identity. Sequence identity can be determined by the BLAST program.
  • an antisense RNA complementary to the transcript of the gene encoding Wee1 or Myt1 or the transcript is specifically cleaved.
  • An RNA having ribozyme activity is also included.
  • RNA may be substituted in part or all by artificial nucleic acids such as PNA, LNA, ENA and the like.
  • these RNAs may be in the form of an expression vector that holds DNA encoding the RNA for expression in cells.
  • those skilled in the art can prepare such RNA by chemical synthesis using a commercially available synthesizer or the like.
  • the “Cdc2 activator” in the present invention may be a compound having an action of suppressing phosphorylation of Cdc2 at these phosphorylated sites. Examples of such a compound include Cdc2 decoy and Cdc2 An antibody is mentioned.
  • Examples of the Cdc2 decoy include a partial polypeptide of Cdc2 containing a site (14th threonine or 15th tyrosine) that is phosphorylated by Wee1 or Myt1.
  • the chain length of such a partial polypeptide is not particularly limited and includes, for example, 5 to 30 amino acids, but does not include the 161st tyrosine, which is a phosphorylation site necessary for activating Cdc2. It is preferable that Such a polypeptide can also be chemically synthesized using a commercially available synthesizer or the like, and can also be prepared using a known recombinant protein production method using Escherichia coli or the like as a host.
  • a cell membrane-permeable peptide may be added to the Cdc2 decoy to increase the efficiency of introduction into the cell.
  • the cell membrane permeable peptide include VP22, Kaposi FGF, TAT, Drosophila Antitinapedia, Penetratin, M918, Transportan-10, Poly-Arginine, and derivatives of these peptides.
  • the cell membrane permeable peptide may be appropriately prepared based on the description of International Publication No. 2007/13255.
  • Examples of antibodies against Cdc2 include antibodies that can specifically recognize and bind to the non-phosphorylated state of the 14th threonine or 15th tyrosine of Cdc2.
  • the form of the antibody may be a polyclonal antibody, a monoclonal antibody, or a functional fragment (nanoantibody, single chain antibody) of these antibodies.
  • Those skilled in the art can appropriately prepare such an antibody by a known antibody production method (hybridoma method, phage display method, etc.) using a partial polypeptide of Cdc2 containing the 14th threonine or the 15th tyrosine. can do.
  • Cdc2 is also activated by dephosphorylation of the 14th threonine or 15th tyrosine of Cdc2 phosphorylated by Wee1 or Myt1 by Cdc25. Therefore, a compound having an action of enhancing the activity of Cdc25 can also be used as the “Cdc2 activator” in the present invention.
  • An example of such a compound is Cdc25. That is, by introducing the protein itself into the cell, the overall activity of Cdc25 in the cell can be enhanced through an increase in the expression level thereof.
  • Cdc25 not only a protein aspect but also an expression vector aspect that holds a DNA encoding Cdc25 in order to express the protein in a cell.
  • the “drug for maintaining monoploidy” of the present invention may contain a physiologically acceptable carrier in addition to the aforementioned Cdc2 activator.
  • physiologically acceptable carriers include isotonic solutions containing physiological isotonic solutions (physiological saline, medium, glucose and other adjuvants (D-sorbitol, D-mannitol, sodium chloride, etc.), etc. ), Excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol, etc.), binders, solubilizers, nonionic surfactants, buffers (phosphate buffers, sodium acetate buffers, etc.) , Preservatives and antioxidants.
  • physiological isotonic solutions containing physiological isotonic solutions (physiological saline, medium, glucose and other adjuvants (D-sorbitol, D-mannitol, sodium chloride, etc.), etc. ), Excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol
  • a transfection reagent for example, a polyamine-based transfection reagent or a cationic lipid-based transfection reagent
  • a transfection reagent may be appropriately added in order to introduce the Cdc2 activator into cells.
  • Example 1 ⁇ Establishment of female parthenogenetic embryo-derived haploid ES cells (phES cells, phESC) and culture thereof>
  • PMSG serum gonadotropin
  • hCG human chorionic gonadotropin
  • Ovulation was induced and unfertilized eggs were collected.
  • the collected unfertilized eggs were treated with strontium chloride (SrCl 2 ) for 30 to 60 minutes, and then cultured in vitro in KSOM medium until they developed in morula.
  • the developed morulae are treated with 1/2 dilution acidic Tyrode to dissolve the zona pellucida, and cultured on ICR mouse-derived feeder cells treated with mitomycin to obtain pure mouse C57BL / 6 Haploid ES cells (phESC-B6) derived from female parthenogenetic embryos were established.
  • the mixed group of phES cells and diploid ES cells prepared in this manner is 20% KSOM, 2 mM L-glutamine, 1 ⁇ non-essential amino acid, 100 uM mercaptoethanol in knockout DMEM. Further, the cells were cultured in a medium in which 3 ⁇ M CHIR99021 and 1 ⁇ M PD0325901 called 2i were further added to 0.1% penicillin / streptomycin, 1000 unit LIF (ESGRO, registered trademark). After 1N cells were collected by FACS, 300 ⁇ M PD166285 was added to the medium and cultured. The mixed group was also cultured in the absence of feeder cells.
  • Cultivation in the absence of feeder cells was performed using a 0.1% gelatin solution in an incubator and treating at 37 ° C. for 30 minutes, then discarding the gelatin solution and using the incubator.
  • Other culture conditions (addition concentration of PD166285, etc.) were the same as those in the presence of the feeder cells described above.
  • 1N cells were collected by FACS by staining with Hoechst 33342 for 30 minutes, washing the cells with PBS ( ⁇ ) with 4% FBS, and then using FACS Aria II (manufactured by Nippon Becton Dickinson).
  • the cells were fixed by treating with 70% ethanol at 4 ° C. for 8 hours or more and analyzed by FACS Calibur (manufactured by Becton Dickinson, Japan) after PI staining.
  • Example 2 ⁇ Establishment of male parthenogenetic embryo-derived haploid ES cells (ahES cells, ahESC) and culture thereof>
  • ahESC male parthenogenetic embryo-derived haploid ES cells
  • ahESC male parthenogenetic embryo-derived haploid ES cells
  • Example 3 ⁇ Establishment of female parthenogenetic embryo-derived epiblast stem cells (phEpiSC, parthenogenetic haploid epiblast stem cell) and culture method thereof> Differentiation induction from phESC established as described above to phEpiSC was performed as follows. That is, differentiation induction into phEpiSC was performed by culturing phESC in DMEM / F12 medium supplemented with N2, B27, NEAA, GlutaMAXI, and ⁇ -mercaptoethanol for 7 days or more.
  • the cells after differentiation induction into phEpiSC are selected from haploid cells using FACS, and then cultured in a medium further supplemented with 20 ng / ml activin A, 20 ng / ml bFGF, 10 nM XAV939 and 300 nM PD166285.
  • 20 ng / ml activin A 20 ng / ml activin A
  • 20 ng / ml bFGF 10 nM XAV939
  • 300 nM PD166285. See Sumi, T. et al., PLoS One, 2013, 8, e63378 for components other than PD166285 regarding the medium in the culture).
  • the cells after differentiation induction into phEpiSC were cultured on feeder cells treated with mitomycin C. Furthermore, in this culture in the presence of PD166285, haploid cells were purified using FACS every 7 days.
  • the haploid phEpiSC was purified by treating with Hoechst at 37 ° C. for 30 minutes, and then selecting only the G1 compartment containing the haploid cells with FACS Aria II. Further, in the differentiation induction from phESC to phEpiSC and the culture after the differentiation induction, the cells are subcultured by treating with complete acactase (registered trademark, complete accurate) at 37 ° C. for 2 minutes. Passage was once at a pace.
  • complete acactase registered trademark, complete accurate
  • the genomic DNA thus prepared was treated with an In-situ oligo DNA microarray kit (manufactured by Agilent) and analyzed by array CGH using 4 ⁇ 180 L slides.
  • the reference genome was labeled with Cy3, and the phESC-derived genome was labeled with Cy5.
  • PCR analysis was also performed using the genomic DNA as a template.
  • anti-Oct3 / 4 antibody goat-derived anti-mouse Oct3 / 4 antibody
  • anti-Nanog antibody rabbit-derived anti-mouse Nanog antibody
  • anti-SSEA1 antibody donkey-derived anti-mouse SSEA1 antibody
  • Alexafluoro 568-conjugated goat-derived anti-rabbit IgG antibody, Alexafluoro 488-conjugated goat-derived anti-mouse IgM antibody and Alexafluoro 488-conjugated donkey-derived anti-goat IgG antibody were each used as secondary antibodies, and the concentration was 1/500. And reacted with phESC at room temperature for 1 hour. These cells were DAPI stained for 5 minutes at room temperature after the secondary antibody reaction. Alkaline phosphatase staining was performed using the SK-5300 kit (manufactured by Vector).
  • teratoma formation 1N cells of haploid ES cells were collected by FACS, and within 7 days, these cells were administered subcutaneously to KSN male mice at 2 ⁇ 10 6 cells / mouse. Then, teratoma was analyzed 30 days after the administration of the cells. The HE staining at the time of analysis was performed according to a conventional method.
  • RNA expression analysis using microarray The gene expression level in phESC obtained by the method of the present invention was analyzed. Specifically, first, 2 ⁇ 10 5 phESC 1N cells and 2N cells of phESC-derived ploidy ES cells were collected by FACS, and total RNA was extracted with Trizol. ES cells established from embryos obtained by natural mating were subjected to RNA extraction without collecting 2N cells by FACS. The RNA thus extracted was treated with a whole mouse genomic DNA oligo microarray kit (manufactured by Agilent) and analyzed with a microarray using 4 ⁇ 44K slides.
  • mice dissected at d14.5 After 14.5 days, or a fetus was obtained by spontaneous delivery and GFP fluorescence was confirmed.
  • the proportion of GFP-positive cells was confirmed for each organ by FACS Aria II. Confirmation of germline migration in chimeric mice was confirmed by detecting GFP expression in fetuses obtained by natural mating of chimeric mice.
  • haploid embryos having one set of maternal genome can be produced by generating parthenogenesis by giving strontium (Sr) stimulation to mouse unfertilized eggs (see Non-Patent Documents 1 to 4). ).
  • the present inventors have partially generated eggs of C57BL / 6 female mice, cultured until in vitro culture until they became morulae, and placed on feeder cells to obtain ES cells (female parthenogenetic embryo-derived 1). It has succeeded in establishing polyploid ES cells (phES cells, phESC).
  • PD166285 an inhibitor against factors (Wee1 and Myt1) that inhibit the transition from G2 phase to M phase in the cell cycle, is used in the culture of haploid ES cell lines. In order to prevent the diploid formation, the culturing was continued for a long time while maintaining the haploid state.
  • phES cells were subcultured 2 to 4 times in a knockout DMEM supplemented with 2i and the like, and then a haploid ES cell line (1N cell) was recovered by FACS and then cultured. Cultivated in the presence (300 nM, 500 nM or 1000 nM) or absence of PD166285, an inhibitor against Wee1 and Myt1. See FIG. 2 for the culturing process, and FIGS. 3 to 6 show the results of FACS analysis in the culturing process. Furthermore, the shape of the cells obtained by culturing in this way and their growth rate were also analyzed. The obtained results are shown in FIGS.
  • FIGS. 3 to 6 it was revealed that a high 1N cell rate can be maintained by recovering the haploid ES cell line by FACS and culturing in the presence of PD166285 from the subsequent culture. Further, as is apparent from the results shown in FIGS. 7 and 8, the cells when 300 nM PD166285 was added to the medium formed more three-dimensional colonies than those not added. Although not shown in the figure, the cell growth rate did not change with or without the inhibitor.
  • the haploid ES cell culture method of the present invention is extremely effective in maintaining the cells for a long time and stably.
  • male parthenogenetic embryo-derived haploid ES cells prepared by the method described in Example 2 in the same manner as the above-described phESC female parthenogenetic embryo-derived haploid ES cells.
  • AhESC male parthenogenetic embryo-derived haploid ES cells
  • histological analysis of the obtained teratomas revealed that three germ layers derived from the injected phESC-B6GFP were formed. Therefore, it was revealed that phESC-B6 established by culturing in the presence of PD166285 maintains undifferentiation and further has differentiation ability in vivo.
  • the method of the present invention it was possible to establish a haploid ES cell line that can be transferred to the germline even with the pure mouse phESC-B6GFP, which is considered to be difficult to establish ES cells and the like. It was revealed that the high quality cell line can be stably maintained.
  • haploid cells differentiated from phESC could be obtained with high efficiency by the method described in Example 3 and FIG. Furthermore, as a result of detecting the expression of the stem cell marker gene in these haploid differentiated cells, as shown in FIG. 22, the expression of Fgf5, a stem cell marker gene unique to EpiSC, was observed in the haploid differentiated cells. On the other hand, the expression of Rex1, a stem cell marker gene unique to ES cells, was not detected in these cells.
  • a differentiated cell can be obtained from a haploid ES cell while maintaining its haploid state, and the diploidy is also maintained in the differentiated cell. It became clear that it was possible.
  • haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability.
  • the present invention it is possible to culture C57BL / 6-derived haploid ES cells while maintaining the haploidity thereof. Therefore, the present invention is extremely useful in forward genetics research and the like.
  • a differentiated cell can be obtained from a haploid ES cell while maintaining its haploidity, and further, the uniploidy can be maintained in the differentiated cell. Can do. Therefore, the present invention is also useful in regenerative medicine, drug discovery development, and the like where there is a strong demand for providing a wide variety of cells, tissues, organs, and the like.

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Abstract

Il a été découvert que, lorsque le gène Cdc2 était activé en continu dans des cellules souches embryonnaires haploïdes, la transition du cycle cellulaire de la phase G2 à la phase M était favorisée et qu'ainsi, l'haploïdie pouvant être conservée. En outre, il a été découvert que les cellules souches embryonnaires haploïdes cultivées ainsi obtenues ne présentaient pas de variations, telles qu'une délétion ou une amplification, dans l'ADN génomique correspondant et, en outre, conservaient la différenciation/pluripotence et l'activité de prolifération élevée. Selon la présente invention, par conséquent, des cellules souches embryonnaires haploïdes peuvent être cultivées tout en conservant l'haploïdie et en conservant la différenciation/pluripotence et l'activité de prolifération élevée, sans provoquer de quelconques variations dans l'ADN génomique.
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WO2012117254A1 (fr) * 2011-03-02 2012-09-07 Cambridge Enterprise Limited Cellules souches embryonnaires haploïdes de mammifère
EP2599859A1 (fr) * 2011-11-30 2013-06-05 IMBA-Institut für Molekulare Biotechnologie GmbH Cellules haploïdes

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EP2599859A1 (fr) * 2011-11-30 2013-06-05 IMBA-Institut für Molekulare Biotechnologie GmbH Cellules haploïdes

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Publication number Priority date Publication date Assignee Title
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